Nanobody-based sandwich reporter system for living cell sensing influenza A virus infection

Abstract

The influenza epidemic is a huge burden to public health. Current influenza vaccines provide limited protection against new variants due to frequent mutation of the virus. The continual emergence of novel variants necessitates the method rapidly monitoring influenza virus infection in experimental systems. Although several replication-competent reporter viruses carrying fluorescent proteins or small luciferase have been generated in previous studies, visualizing influenza virus infection via such strategy requires reverse genetic modification for each viral strain which is usually time-consuming and inconvenient. Here, we created a novel influenza A nucleoprotein (NP) dependent reporter gene transcription activation module using NP-specific nanobodies. Our results demonstrated the modular design allowed reporter genes (mNeonGreen fluorescent protein and Gaussia luciferase) specifically expressing to detect intracellular NP protein, and therefore acts as a universal biosensor to monitor infection of various influenza A subtypes in living cells. The new system may provide a powerful tool to analyze influenza A infections at the cellular level to facilitate new antiviral drug discovery. Moreover, this approach may easily extend to develop live-cell biosensors for other viruses.

Influence of nanobody binding on fluorescence emission, mobility, and organization of GFP-tagged proteins

“…An explanation for these observations could be that the (E)GFP tagged proteins appear to a certain extend in aggregates or homo-oligomers that are (partially) disassembled after Nb binding, leading to an average increase in fluorescent particle number (Figures 2 and 3) and mobility ( Figures 3 and 4) without majorly affecting the fluorescence lifetime ( Figure S3). GPI-EGFP dimers or GFP oligomers have been reported previously (Aronson et al, 2011;Huang et al, 2015;Jain et al, 2001;Suzuki et al, 2012a) and could be mediated by the FP tag itself (Beutel et al, 2015;Zacharias et al, 2002;Wang et al, 2019). However, since aggregates should in principle be brighter, one would in this case expect a decrease in molecular fluorescence brightness (cpm) upon aggregate disassembly.…”

“…Here, we only showed the effect of one specific anti-GFP nanobody (GFP-booster from Chromotek) and we also acknowledge that we only tested one GFP and one EGFP variant expressed on the surface of the living cells (see Transparent methods for protein sequences). Many derivatives of GFP exist that counteract FPmediated oligomerization especially when passing through oxidative environments (Aronson et al, 2011;Zacharias et al, 2002;Suzuki et al, 2012b;Wang et al, 2019). An interesting approach could be to add a short tag to the FP to allow detection with another nanobody (Braun et al, 2016).…”

“…Nanobodies have successfully been raised against various target molecules and used in microscopy ( Pleiner et al., 2015 , 2018 ). Some examples for nanobody epitopes include histones ( Jullien et al., 2016 ), viral proteins ( Cao et al., 2019 ), artificial peptides ( Braun et al., 2016 ), clathrin coat components ( Traub, 2019 ), vimentin ( Maier et al., 2015 ) and many more ( Aguilar et al., 2019 ; Mikhaylova et al., 2015 ). Interestingly, a study using nanobodies targeting synaptic proteins and making use of the nanobodies' smaller size and better penetration capabilities suggested a new pool of synaptic vesicles ( Maidorn et al., 2019 ).…”

Broad Reactivity Single Domain Antibodies against Influenza Virus and Their Applications to Vaccine Potency Testing and Immunotherapy

“…In a different approach, an engineered sdAb with a mouse IgE-Fc was used in a cell-based sensor; again, no data on breadth of reactivity and applicability was provided [ 134 ]. Another cell sensor-type assay used two sdAbs against the viral NP; one sdAb was expressed in the cell as a fusion protein with a DNA-binding domain, the other was fused to a transcription activation domain; upon infection of the cell with influenza virus, NP acted as a scaffold to assemble a bipartite transcription factor, leading to the expression of a reporter gene [ 135 ]. Due to the conserved nature of the NP, this system was able to detect viruses of three subtypes (H3, H5, H7).…”

Influence of nanobody binding on fluorescence emission, mobility and organization of GFP-tagged proteins

“…Nanobodies have successfully been raised against various target molecules and used in microscopy (Pleiner et al, 2015(Pleiner et al, , 2018. Some examples for nanobody epitopes include histones (Jullien et al, 2016), viral protein (Cao et al, 2019), artificial peptides (Braun et al, 2016), clathrin coat components (Traub, 2019), vimentin (Maier et al, 2015) and many more (Aguilar et al, 2019;Mikhaylova et al, 2015).…”